Chemical analysis of water, foodstuffs like milk and biological fluids such as serum and urine is often desirable or necessary. Various analytical elements to facilitate such analyses are known. Generally, such elements include a reagent (hereinafter termed interactive composition), for a substance under analysis (hereinafter termed analyte). The interactive composition, upon contact with a liquid sample containing the analyte, effects a detectable change in response to the presence of the analyte. For example, such a detectable change can be the formation or disappearance (e.g. reduction) of a detectable species, e.g. a dye. Such a change can be determined as it occurs (i.e. a rate assay), or after a certain time (i.e. endpoint assay).
In the diagnostic clinical analysis of complex biological fluids, such as serum, whole blood, urine, etc., it is known that substances present in the patient sample other than the analyte being determined, can interfere with or bias the analytical results such that the end results are not in direct proportion to the concentration of the analyte. This interference is particularly harmful in analyses of analytes which are present in the sample at low concentrations (e.g. certain low level enzymes). Such interference is also particularly prominent when the analyte is determined using a rate assay versus an endpoint assay.
For example, in the analysis of two low level enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), using conventional NADH-based interactive compositions, the rate of enzyme activity in a sample is determined by observing the rate of disappearance of NADH which can be spectrophotometrically measured at 340 nm. It has been found that the results of such AST and ALT analyses are adversely affected by the presence of bilirubin and proteins in the liquid test sample. In the presence of bilirubin, the results were positively biased away from the true analyte concentration. It was also observed that variations in total protein concentration altered the results such that AST and ALT were underestimated at low protein levels and overestimated at high protein levels.
Recently, much work has been directed toward developing dry analytical elements useful in diagnostic chemical analysis, where testing of biological fluids provide highly quantitative results quickly and conveniently. For example, U.S. Pat. No. 3,992,158 (issued Nov. 16, 1976 to Przybylowicz et al) describes integral analytical elements which are a significant advance in the clinical chemistry art. However, such elements need further modification to overcome the interference of bilirubin and protein noted above and to provide improved assay accuracy.
In U.S. Pat. No. 4,153,668 (issued May 8, 1979 to Hill et al), immobilized polymeric cationic materials are incorporated into the spreading layer of dry analytical elements in order to more uniformly disperse a liquid sample of a negatively-charged protein-bound or proteinaceous substance. This reference teaches that it is essential to the described advantage of uniform distribution of protein analyte that the cationic material be immobilized so that it cannot migrate throughout the element in order to assay for proteins. The purpose of incorporating the charged polymers is to uniformly disperse the liquid sample and to reduce a chromatographic effect. There is no teaching that the charged polymers remove interfering materials, such as bilirubin or proteins.
Other immobilized, positively charged polymeric materials are described in U.S. Pat. No. 4,069,017 (issued Jan. 17, 1978 to Wu et al). Such materials are used to mordant bilirubin in hydrophilic layers beneath a spreading layer in elements adapted for the analysis of bilirubin. It has been observed that such materials fail to consistently remove the effects of bilirubin or protein interference.
U.K. patent application No. 2,085,581 (published Apr. 28, 1982), teaches the use of tertiary amines in hydrophilic extraction layers of elements designed for the determination of bilirubin. Such amines apparently perform similarly to polymeric mordants in a separate extracting layer.
Nowhere does the art teach how one skilled in the clinical chemistry art can perform assays using dry elements for analytes other than bilirubin or proteins whereby the interfering effects of bilirubin or protein are avoided. Hence, there is a need in the art for a dry analytical element which is capable of providing highly accurate analyses of liquid samples and of avoiding the interfering effects of bilirubin or proteins in the liquid sample.